Method and system for magnetic extraction of components in a liquid sample

ABSTRACT

A system for extracting analytes from a biological sample, which includes: an electronic pipette having pipette cones with a tip; a well support; a pipette holder including: a base which can removably house each well support; a pipette support into which the pipette is inserted, and which can move relative to the base between a first position in which the tips of the cones are inserted in a well of the support and at least one second position in which the tips are outside the wells; a housing facing the pipette cones above their tip when the pipette support is in the first position, and facing the tips of the pipette cones when the pipette support is in the second position; and a magnetized part removably inserted in the housing.

FIELD OF THE INVENTION

The invention relates to the field of the extraction of componentscontained in a solution using magnetic particles.

The invention can in particular be used in the biological samplepreparation field, in particular in the implementing of in vitrodiagnosis, by capturing analytes of biological origin (nucleic acids,microorganisms, proteins, peptides, etc) present in a solution.

PRIOR ART

Originally developed for the extraction of nucleic acids present in abiological sample and described in document U.S. Pat. No. 5,234,809, the“BOOM®” technology consists in introducing, into a liquid sample,magnetic particles capable of binding with components of interest, thenin separating the magnetic particles from the sample by means of one ormore magnets. The particles thus captured can then undergo a subsequenttreatment, for example to release their components into a recoverysolution. Because of the efficiency of this technique, many devices havebeen developed and marketed, in particular for DNA and RNA, etc.), thesebeing both manual devices (for example the NucliSENS-miniMAG® from theapplicant) and automated devices (NucliSENS-easyMAG® from theapplicant). However, these automated devices have various limitations.

A first limitation concerns their polyvalence and their bulkiness.Indeed, these devices are usually heavy and bulky automated deviceswhich are designed for carrying out a sequence of treatments that cannotbe modified by the user. An automated device is thus designed for asingle type of extraction, for example designed for the purification ofnucleic acids, but incapable of carrying out a magneticimmunoconcentration.

A second limitation concerns the circuits for injecting and suctioningthe various liquids used during the extraction. Since the number ofliquids is high, this implies circuits which are also numerous and/orcomplex. Furthermore, because of possible contaminations, theseinjection/suction circuits must be regularly cleaned, which impliestaking the devices out of service.

A third limitation concerns the stirring operations which are carriedout in order to obtain homogeneity of the sample comprising the magneticparticles before capture thereof, in order to maximize the capture bysaid particles of the analytes of interest or in order to efficientlywash the magnetic particles. This type of stirring usually requirescomplex mechanisms, for example based on mobile magnets which cause themagnetic particles to move.

The fourth limitation concerns the various liquids used during theextraction. Usually, the steps carried out for the extraction areperformed in or starting from a single container. As a result, thiscontainer fixes an identical volume for all the liquids involved (e.g.the sample, the various washing solutions, the eluting solution, etc),which limits the overall efficiency of the extraction process. Indeed,some treatments (e.g. washing) require large volumes in order to becompletely efficient, whereas other treatments only need a small volumeof liquid (e.g. the elution).

SUMMARY OF THE INVENTION

The objective of the present invention is to provide a method forextraction of components in a liquid sample by means of magneticparticles which offers great freedom in the choice of the liquidvolumes, in particular up to 10 ml, used during the extraction.

To this effect, a subject of the invention is a method for extractingcomponents contained in a biological sample in liquid form, saidcomponents being capable of binding to magnetic particles, the methodcomprising:

-   -   a phase of mixing the sample with the magnetic particles;    -   a phase of suctioning the mixture from a well in a tubular        pipette cone comprising a tip intended for pipetting liquid;    -   a phase of capturing the magnetic particles on an internal wall        of the pipette cone:        -   by applying a first magnetic field to the pipette cone, said            field being capable of attracting and holding the magnetic            particles in a predetermined zone of the pipette cone,            termed “capture” zone, above the tip of said cone;        -   and by applying at least one cycle of suction and discharge            of the mixture contained in the pipette cone in a well;    -   at least one phase of washing the particles captured on the        internal wall of the pipette cone by:        -   discharging the mixture contained in the pipette cone; and        -   applying, from a well containing a washing solution, at            least one cycle of suction and discharge of the washing            solution in the pipette cone;    -   a phase of migration of the magnetic particles on the internal        wall of the cone, from the capture zone to the tip of the        pipette cone, by carrying out a relative movement of the pipette        cone relative to the first magnetic field;    -   and a phase of transferring said magnetic particles having        migrated into the tip of the pipette cone into a recovery well        containing a solution.

In other words, the invention takes advantage of a pipette cone in whichsuction and discharge cycles can be carried out while dipping the tip ofsaid pipette cone into a well. By virtue of such cycles, it is possibleto capture all of the particles present in a sample of much greatervolume than that of the volume, i.e. the cone, in which the extractionis carried out. It is even possible to pass through the cone acumulative volume of liquid much greater than the volume of the sampleitself, by regulating the number of suction and discharge cycles.Likewise, the volume of the washing solution(s) used, where appropriate,during the extraction can be much greater than the volume of the cone.The migration phase makes it possible, for its part, to localize themagnetic particles in a portion of the cone, the tip, which can dip intoa well of very small volume. The volume of the recovery solution cantherefore be small if necessary. The volume of the recovery solution isthus independent of the volume of the sample and of the volume of thepipette cone in which the extraction is carried out. By virtue of theinvention, it is consequently possible to optimize each volume of liquidused, and thus to optimize the extraction.

Furthermore, because of the geometry of the tube-shaped cones and of thesuction/discharge cycles, efficient stirring of the sample in the cones,said stirring being for example carried out before the capture, and alsoefficient washing, are obtained, this being without recourse tomechanisms of mobile magnet type. In addition, the inventors have notedthat efficient washing is obtained in the cone even though the particlesare captured on the wall of the pipette cone. A large volume of washingsolution can be used, further increasing the efficiency of the washing.As a result, all the steps of the extraction (stirring, capture,washing, transfer into a recovery solution) can be carried out in thepipette cone.

According to one embodiment, the movement of the first magnetic fieldconsists in moving the pipette cone parallel to a longitudinal axis ofsaid cone, and in keeping the first magnetic field constant, thelongitudinal axis of the pipette cone remaining at an equal distancefrom the first magnetic field during the movement of the pipette cone.

In other words, the migration of the particles can be carried out simplyby moving the pipette cone relative, for example, to a permanent magnet.

According to one embodiment, the transferring phase comprises:

-   -   placing the tip of the pipette cone in the recovery well;    -   and applying a second magnetic field from the bottom of the        recovery well so as to cause the magnetic particles contained in        the tip of the pipette cone to migrate into the recovery well.

In particular, the second magnetic field is produced by a magnetpositioned partially or entirely under the tip of the pipette cone. Thefirst magnetic field applied to the pipette cone is deactivated duringthe application of the second magnetic field.

In other words, the second magnetic field makes it possible to simplyattract the particles into the recovery well, which increases the speedof recovery of the magnetic particles in the recovery well, and also thenumber of particles recovered. Furthermore, the second magnetic fieldautomatically captures the magnetic particles in the recovery well. Forexample, if the recovery solution is an eluent, the components bound tothe particles have been released and a technician can directly pipettethe solution which is free of magnetic particles.

According to one preferred variant, the transferring phase comprises thedeactivation of the first magnetic field followed by the application ofcycles of suction and discharge of the solution of the recovery well inthe tip of the pipette cone, said application comprising:

-   -   a first phase of applying the cycles at a first frequency;    -   followed by a second phase of applying the cycles at a second        frequency, lower than the first frequency.

The first phase makes it possible to efficiently disaggregate the clumpsof particles captured on the pipette cone, also called “pellet”, andthus to resuspend the particles in the recovery solution. The secondphase makes it possible to continue to stir the solution while at thesame time not opposing the migration of the particles under the effectof the magnetic field. This makes it possible to increase even furtherthe speed of recovery and the number of particles recovered in therecovery well. Furthermore, if the solution is an eluent, the functionof which is to release the components captured by the magneticparticles, these cycles have the effect of stirring the particles in theeluent, which increases the efficiency of the eluent, in particular whenan eluting solution is used in the detaching of the analytes from themagnetic particles without a heating step.

According to one embodiment, the method comprises, prior to thecapturing phase, a phase of stirring the mixture contained in thepipette cone by applying at least one cycle of suction and discharge ofsaid mixture in the pipette cone. Because of the geometry of the cone,which is tubular in shape, it is possible to obtain a high volumethroughput relative to the cross section of the cone, and consequentlyefficient stirring. Furthermore, high turbulences exist in the cone thatare naturally generated by the flow of the liquid, said turbulencesincreasing the efficiency of the stirring. Advantageously, a disposableaccessory is provided in the cone for increasing this effect.

According to one embodiment, the method comprises, prior to thetransferring phase, at least one phase of washing the particles capturedon the internal wall of the pipette cone by:

-   -   deactivating the magnetic field;    -   releasing the captured particles by applying, from a well        containing a washing solution, at least one cycle of suction and        discharge of the washing solution in the pipette cone;    -   applying a second phase of capturing on an internal wall of the        pipette cone:        -   by applying the first magnetic field to the pipette cone,        -   and by applying at least one cycle of suction and discharge            of the mixture contained in the pipette cone in the well            containing the washing solution.

According to one embodiment, the release of the captured particlescomprises a phase of applying the cycles in such a way as to carry outan up and down movement of a meniscus of said solution over a pellet ofparticles captured in the pipette cone, said up and down movement ofsaid meniscus being carried out on a portion of the cone less than thetotal length of the pipette cone.

More particularly, the release of the captured particles comprises asecond phase of applying the cycles in such a way as to totally suctionand discharge the cone washing solution. The frequency of application ofthe cycles of the second phase is lower than the frequency ofapplication of the cycles of the first phase.

In particular, prior to the release of the captured particles, themethod comprises at least two washing phases carried out in two distinctwashing solutions.

This embodiment is particularly advantageous when the componentscontained in the biological sample are nucleic acids (e.g. DNA, RNA).

According to another embodiment, the components contained in thebiological sample are microorganisms (e.g. bacteria, fungi, yeasts), andthe method comprises a single capturing phase and a single washingphase.

In particular, the mixture of the sample with the magnetic particles hasa volume of greater than 1 milliliter, and preferably greater than orequal to 2 milliliters, and the volume of the recovery well is less thanor equal to 200 microliters, and preferably less than or equal to 100microliters.

According to one embodiment, the method comprises, prior to thetransferring phase, at least one phase of washing the particles capturedon the internal wall of the pipette cone:

-   -   by suctioning the washing solution in said pipette cone;    -   then by modulating the first magnetic field applied to the        magnetic particles in order to capture said particles on the        internal wall of the pipette cone;    -   then by discharging the pipette cone washing liquid.

In other words, the modulation of the magnetic field induces areorganization of the pellet of particles captured on the wall of thecone. In particular, the pellet can change shape, or can spread, slip orelse “roll” on the wall of the pipette cone. This reorganization of thepellet makes it possible to further increase the efficiency of thewashing, this being all the more since this reorganization can becarried out together with cycles of suction and discharge of the washingsolution.

In particular, the modulation of the first magnetic field is carriedout:

-   -   by moving the pipette cone parallel to a longitudinal axis of        said cone, and by keeping the first magnetic field constant;    -   and/or by passing magnets spaced out from one another in front        of the captured particles.

In other words, the modulation is obtained simply, for example by atechnician who slides a strip of magnets or by an automated device whichapplies a simple mechanism of translational movement of a strip ofmagnets.

According to one embodiment, the volume of the pipette cone is at leastten times greater than the volume of the recovery well. According to oneembodiment, the volume of the mixture is at least three times greaterthan the volume of the pipette cone.

According to one embodiment, the components belong to the group formedby single-stranded or double-stranded nucleic acids (DNA and/or RNA),microorganisms, proteins and peptides. The components consist of anyother type of molecules depending on the functionalization given to themagnetic particles.

The aim of the present invention is also to provide a device forcarrying out the method which has just been described, which is not verybulky and which is simple for a laboratory technician to use.

To this effect, a subject of the invention is also a pipette holdercomprising:

-   -   a base;    -   a recess made in the base, which can removably house a well        support;    -   a pipette support comprising a first housing into which can be,        advantageously removably, inserted a pipette equipped with at        least one tubular pipette cone comprising a tip intended for        pipetting liquid, the first housing opening out onto the recess        of the base, the pipette support being translationally mobile        relative to the base in a direction parallel to an axis of the        pipette cones and mobile between a first position in which the        tip of each pipette cone is inserted in a well of the well        support and at least one second position in which said tip is        outside said well;    -   a second housing which can removably house a magnetized part,        the second housing facing each of the pipette cones in a        position above the tip thereof when the pipette support is in        the first position, and the second housing facing the tip of the        pipette cone when the pipette support is in the second position.

In other words, the pipette holder receives a pipette and the techniciancarries out the steps of the extraction method by raising/lowering thepipette, in particular to cause migration of the particle pellet in thetip of the pipette cone(s), by introducing wells (in the form of aplate, a strip, etc.) into the base, and by actuating the pipette.

The pipette holder, which is not very bulky and is transportable, alsoallows semi-automation of the extraction method when an electronicpipette is used. Such a pipette in fact comprises circuits for suctionand discharge in each pipette cone equipping it, and amicroprocessor-based electronic circuit. This electronic circuitcontrols the suction/discharge circuits as a function of setpointsentered by the technician by means of an interface equipping the pipetteand/or of a computer/tablet/smartphone connected to the pipette (e.g. bya wireless connection of Bluetooth type), etc. These setpoints consistfor example of suction/discharge cycle instructions and/or of a choiceof a particular protocol prerecorded in the pipette.

Since the electronic pipette is programmable, a great deal ofpolyvalence is also obtained in the definition of the extraction method,which can be adjusted as a function of a desired particular magneticcapture (e.g.: nucleic acid purification, magnetic immunoconcentration,etc.). In particular, a protocol suitable for the intended extractioncan be recorded in the pipette, the protocol being defined in terms ofnumber of suction and discharge cycles, of cycle sequence, of cyclefrequency, of time between the cycles, defined volumes, etc. Anautonomous and semi-automated system is thus obtained.

Finally, the pipette cones are detachable from the pipette, andtherefore easily replaceable, without the pipette being taken out ofservice for a long period of time.

According to one embodiment, the first housing comprises an opening forthe frontal insertion of the pipette into and the frontal removal of thepipette from the first housing of the pipette support. The frontalinsertion and removal of the pipette and of the cones in positionminimize the risk of touching the pipette holder with the cone tips, andtherefore the risk of contamination of the pipette holder.

According to one embodiment, the second housing is made in the base.

In particular, the pipette support comprises a third housing into whichthe magnetized part can be removably inserted in order to face eachpipette cone at a position above the tip of said cone when the pipettesupport is in the second position.

In other words, when the magnetized part (e.g. comprising one or morepermanent magnets) is present in the third housing, it is rigidly linkedto the pipette cones and therefore follows their translational movementrelative to the base. During such movements, the magnetized parttherefore keeps the pellets of magnetic particles attached in the cones.The technician can thus for example raise the pipette in order to moreeasily move a well support in the base without the risk of moving theparticle pellets in the cones.

According to one particular embodiment, the second and third housingscommunicate, and the pipette support comprises means capable ofremovably maintaining the magnetized part in the third housing. In thisway, the technician can detach the magnetized part from the pipettesupport which then automatically takes a place in the base by fallinginto the second housing. This detachment takes place in particular forthe operation of migration of the magnetic particles in the cone tips.

According to one embodiment:

-   -   the base comprises at least one toothed wheel which can rotate;    -   and the pipette support comprises a rack which engages with the        toothed wheel in order to translationally move the pipette        support relative to the base during the rotation of the toothed        wheel.

In particular, the pipette holder comprises a device for locking andunlocking the pipette support in the first position. In particular, thepipette holder comprises at least one handle that is rigidly connectedto the toothed wheel so as to turn said wheel and that is capable ofremovably attaching to a handle that is rigidly connected to the toothedwheel of another pipette holder, which therefore makes it possible toincrease the number of pipette cones during the extraction method.

The subject of the invention is also a system for extracting componentscontained in a biological sample in liquid form, said components beingcapable of binding to magnetic particles, the system comprising:

-   -   a pipette equipped with at least one tubular pipette cone        comprising a tip intended for pipetting liquid and with a        circuit for suction and discharge in each pipette cone;    -   at least one well support;    -   a pipette holder comprising:        -   a base;        -   a recess made in the base, which can removably receive each            well support;        -   a pipette support comprising a first housing into which the            pipette is inserted, advantageously removably, the first            housing opening out onto the recess of the base, the pipette            support being translationally mobile relative to the base in            a direction parallel to an axis of the pipette cones and            mobile between a first position in which the tip of each            pipette cone is inserted in a well of the well support and            at least one second position in which said tip is outside            said well;        -   a second housing facing each of the pipette cones in a            position above the tip thereof when the pipette support is            in the first position and the second housing facing the tip            of the pipette cone when the pipette support is in the            second position; and    -   a magnetized part removably inserted in the second housing.

In particular, the pipette holder is in accordance with the pipetteholder described above.

An aim of the invention is also to provide a well support for themigration of magnetic particles from the tips of pipette cones intorecovery wells.

To this effect, a subject of the invention is also a well support,comprising a part in which recesses for receiving the wells are made,and at least one magnet facing each of the recesses made in said part.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be understood more clearly on reading the descriptionwhich follows, given solely by way of example, and made in relation tothe appended drawings, in which identical references denote identicalelements, and in which:

FIG. 1 is a perspective view of an extraction system according to theinvention;

FIGS. 2A and 2B are face-on perspective views of an electronic pipetteand of the removable pipette cones thereof;

FIGS. 3A and 3B are face-on perspective views of a pipette holderaccording to the invention;

FIGS. 4A and 4B are detailed sectional views of the pipette holder ofFIG. 3 , respectively along the planes A-A and B-B of FIG. 3B;

FIG. 5 is a perspective view of a “deepwell” plate comprising wells;

FIGS. 6A and 6B are perspective views of a magnetic rack and of PCRelution tubes that can insert into the rack;

FIG. 7 is a face-on view of a magnetized part according to theinvention;

FIG. 8 is a flowchart of an extraction method according to theinvention;

FIG. 9 is a photograph of the pipette cones of a system according to theinvention with pellets of magnetic particles placed approximatelyhalfway up the cones;

FIG. 10 is a photograph of these same cones with the pellets placed inthe tips of said cones;

FIG. 11 is a photograph of PCR elution tubes in which the magneticparticles have been recovered;

FIGS. 12 and 13 illustrate a second embodiment of the pipette holderaccording to the invention;

FIG. 14 is a perspective view of two systems of FIG. 1 , coupled to therotation handle means;

FIG. 15 is a diagrammatic view illustrating the up and down movement ofa meniscus of a solution on a pellet of particles for detaching saidpellet from the wall of a pipette cone.

Except for FIG. 15 , the description is given in relation to planes andphotographs on a reduced scale of an actual system.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIGS. 1 to 7 , a system 10 for extracting (FIG. 1 )components contained in a liquid sample comprises an electronic pipette12 (FIG. 2 ), a pipette holder 14 (FIG. 3 ) into which the pipette 12 isinserted, one or more well supports 18 a, 18 b (FIGS. 5 and 6 ) whichcan each be inserted into the pipette holder 14, and a first magnetizedpart 16 (FIG. 7 ).

The pipette 12, which is portable, comprises a row of pipette cones 20,and a body 22 on which the cones 20 are mounted (FIG. 2A). This body 20houses a circuit for suction/discharge of liquid in the cones 20 (forexample a set of pistons actuated by an electric motor), and anelectronic circuit for controlling the suction/discharge circuit. Theelectronic circuit, which comprises for example a microprocessor and oneor more computer memories, is programmable, and has, embedded within it,instructions for carrying out one or more pipetting protocols, eachprotocol comprising one or more steps. The electronic circuit alsocomprises a man-machine interface 24 housed in a handle 26 of the body22, the interface comprising a set of selection and navigation buttons28 and a display screen 30 allowing visualization and selection ofvarious recorded pipetting protocols. The user can in particular programthe electronic pipette 12, e.g. by downloading, into said pipette,instructions from a computer connected to the pipette 12 through awireless link, for example Bluetooth. The user can also select, via theinterface 24, a prerecorded protocol. The pipette 12 is moreover capableof suctioning a predefined volume of liquid into each of the cones 20,discharging a predefined volume from each of the cones, and carrying outautomatic suction/discharge cycles of variable time and frequency.

As illustrated more particularly in FIG. 2B, the cones 20, which aretubular in shape and have a longitudinal axis X, are removable byfitting to lugs 32 protruding from the body 22, which allows them to bereplaced. The cones consist moreover of a plastic, for example apolypropylene, which has the effect of making them “transparent” to amagnetic field and allows the capture of magnetic particles, as willsubsequently be described in greater detail. Finally, each cone 20 has atapered profile 21, or “tip”, at its open pipetting end. This part 21has a reduced cross section in a plane perpendicular to the axis X,which makes it easier to introduce it into containers or wells, as isknown per se.

The electronic pipette 12 is for example the “8-channel Viaflo II” modelsold by the company ©Integra Biosciences AG, Switzerland, the elementsof which model are described in patent applications US 2009/071266, US2009/074622, US 2011/076205 and US 2008/095671.

The pipette holder 14 for its part comprises (FIGS. 3A and 3B) a base34, intended to be placed on a workbench (e.g. a laboratory table orbench), and also a mobile part 36 which moves relative to the base 34.The mobile part 36, also called “pipette support”, comprises a housing38 into which the pipette 12 can be removably inserted and kept immobileas illustrated in FIG. 1 . For the translational movement of the pipettesupport 36 relative to the base 34, the support 36 comprises one or moreracks 40, for example two of them, which engage with toothed wheels 42mounted on an axle 44 which rotates and which is inserted in the base34. One or more rods 46 are also attached to the support 36(respectively in the base 34) and slide in orifices of the base 34(respectively in the support 36) in order to guide the support 36 in itstranslational movement. One or two rotation handles 50 are also attachedto the end of the axle 44 in order to allow the user to easily turn thelatter in the direction of the arrows 52 (FIG. 1 ) and therefore causethe pipette support 36 to rise and descend as illustrated by the arrows54 (FIG. 1 ). The translational movement of the pipette support 36relative to the base 34 is thus parallel to the axis X of the cones 20when the pipette 12 is placed in the housing 38 of the support 36, andtherefore parallel to the direction of gravity when the base 34 isplaced on a horizontal workbench, such that the support 36 “rises” or“descends”.

The base 34 is open on its front face 56 so as to allow the introductionand the removal of the well supports 18 a, 18 b, thus defining a housingfor the latter. This housing is open on its upper part so as to allowthe cones 20 of the pipette 12 to reach said well supports when thepipette descends. Thus, as described in greater detail below, thepipette 12 can take up several positions relative to the base 34, andthus relative to a well support 18 a, 18 b inserted in the latter. Inparticular, the pipette 12 can take up a position in which the tips 21of the cones 20 dip into wells of the support 18 a, 18 b, and at leastone position in which the tips 21 do not dip into the wells, and are ata distance from the latter so as to allow the well supports to behandled by the user and magnetic particles to be captured in a centralposition of the cones 20.

With reference to FIGS. 5 and 6 , the well supports can have severalforms depending on the desired extraction. In particular, a well supportis a compartmentalized plate 18 a (FIG. 5 ), usually called a“microplate” of “DeepWell” type. This type of plate comprises rows 60 ofwells 52 into which the row of pipette cones 20 can dip. Each row 60 canthus receive a particular liquid used during the extraction step carriedout by the system 10. The passing from one row 60 to the other is thencarried out simply by the user who places, in line with the pipettecones 20, the particular row 60 containing the liquid required for thestep that has to be carried out.

Another well support, described in FIG. 6A, is magnetized andspecifically designed for the migration of magnetic particles from thetips 21 of the pipette cones 20 into wells. The support 18 b comprisesfor this purpose a body 64 in which is made a row of housings 66 thatcan receive the row of pipette cones 20, and in which is inserted asecond magnetized part 68 comprising one or more permanent magnets, forexample a permanent magnet in proximity to each of the wells 66. Themagnetized part 68 is placed under the wells 66 or facing their lowerportions as illustrated. Thus, the tips 21 of the pipette cones 20 areplaced above the magnetized part 68 when said tips are dipped into thewells 66. Finally, the support 18 b serves as a magnetic rack whichremovably receives tubes 69 in the housings 66, for example PCR elutiontubes as illustrated in FIG. 6B, for the purposes for example ofsubsequent transfer of the extraction product recovered therein.

The first magnetized part 16, the function of which is to capturemagnetized particles in the cones 20 in a manner subsequently describedin greater detail, comprises, for its part, one or more permanentmagnets 72, advantageously a row of permanent magnets separated from oneanother by spaces 74, and even more advantageously a permanent magnetfacing each pipette cone 20 when the part 16 is entirely inserted in thebase 34. The part 16 also comprises a handle 76 for better gripping bythe user.

A housing 78 for receiving the magnetized part 16 is provided in thebase 34, the housing 78 being placed such that the part 16 faces thepipette cones 20 above their tip 21, and preferably faces a central zone80 at a height greater than the well, when the tips 21 dip into thewells, held in a well support. In this way, the particles are capturedin a volume of the cone that is sufficiently large so as not to formplugs in the cones.

The pipette holder 14 also comprises means for controlling the speed atwhich the support 36 rises. In particular, the rack and the toothedwheel are designed such that half a turn (180°) of the wheel 50 makes itpossible to travel across the whole of the rack, and a flyweight 58integrated into each of the handles 50 in an off-axis manner relative tothe axle 44. These flyweights, under their weight and the associatedlever effect, generate a rotation couple which rotates the axle 44 whileat the same time limiting the couple transmitted by hand by the user.Advantageously, as illustrated in FIG. 4A, a substantial part of theaxle 44 is also formed of a semi-cylindrical flyweight for the samepurpose. This mechanical assistance helps to raise the pipette support,and therefore limits musculoskeletal problems, and carries out brakingwhich allows the user to more accurately control the speed at which thesupport 36 rises and descends.

Other mechanisms for controlling the speed of the support 36 can beprovided for, in particular magnetic braking. For example, withreference to FIG. 4A, the flyweight 59 comprises a magnetizable material(e.g. steel or equivalent) and a third magnetized part 80(parallelepipedal or in the shape of an arc of a circle concentric tothe axle 44) is housed in the base 34, preferably facing the magnetizedpart 16 with respect to the axle 44 so as not to disrupt the extraction.When the flyweight 59 of the axle passes in front of the magnetized part80, the rotational movement of the axle 44 is slowed because of thebraking couple generated. This makes it possible, on the one hand, tocompensate for the effort for raising the pipette during the rise (byacting as assistance for the user) and, on the other hand, to controlthe speed at which the pipette rises when it is desired to cause themagnetic particles to descend to the bottom of the pipette cones, aswill be described below.

A stop mechanism is also advantageously provided for, as illustrated inFIG. 4B. In this variant, a wheel 84 made of deformable material (e.g.of elastomer) is mounted on the axle 44 and comprises two teeth 86, 88.A protuberance 82, for example a hemispherical protuberance, moreoverprotrudes from the base 84 facing the wheel 84. When the user raises thepipette 12 by actuating the wheel 50, the first tooth 86 encounters theprotuberance 82. When the cut applied to the wheel 50 is increased, thetooth 86 bends, passes the protuberance 82, and returns to its shape. Inthis position, the tooth 86 can then rest on the protuberance 82, thehardness of this tooth being chosen such that it does not bend under theaction of the weight of the pipette 12 and of the pipette holder 36. Thepipette is thus blocked in the top position, the user thus being able torelease the wheel 50. The second tooth 88, which is larger in dimension(e.g. length and/or width) requires a much greater couple in order forit to pass, and thus defines a stop for preventing the pipette holder 36from disconnecting from the base 34, unless the user deploys a forcecapable of breaking this tooth. As a variant, the protuberance 82 isreplaced by a stop comprising a ball mounted on a spring in a housing ofthe base. The wheel 84 can thus be made of a hard material. The actionof the first tooth 86 then has the effect of pushing the ball into itshousing, thus allowing the tooth 86 to pass.

Presently described is a method for extracting components contained in aliquid sample by means of magnetic particles, this method being carriedout by means of the system which has just been described. The method isbased on the combination of the pipette holder, the programmableelectronic pipette and pipette cones (e.g. with a volume of 1250 μl) inorder to carry out the various steps of capturing, washing and elutingmagnetic particles for treating a sample volume per pipette cone ofbetween 1 ml and 5 ml. The capture of the magnetic particles is carriedout sequentially in the pipette cones during suction/discharge cycles onall of the volume of the sample to be treated. By way of example, amethod for purifying viral nucleic acids using NucliSENS© chemistry,namely an extraction of nucleic acids by means of magnetic silicaparticles, is described in relation to the flowchart of FIG. 8 .

The method begins with a step 100 of preparing the various samples andreagents required for the purification, followed by said purification in102.

In particular, the preparation 100 consists, in 104, in mixing thebiological sample comprising viruses from which it is desired to extractthe nucleic acids, with a reagent for chemical lysis of viruses (e.g.the “Nuclisens miniMAG” lysis reagent from bioMérieux, reference 200292,or the “Nuclisens easyMAG” lysis reagent from bioMérieux, reference280130), in a proportion of two volumes of lysis reagent for one volumeof sample. The mixture is then heated for 30 minutes at 56° C., thusreleasing the nucleic acids from the viruses in a manner known per se.Magnetic silica particles (e.g. particles having a paramagnetic,ferromagnetic or ferrimagnetic core which may or may not exhibitremanence, said core being covered with a silica shell), having theproperty of binding with nucleic acids, are then introduced, in 106,into the lysed sample.

The preparation 100 continues, in 108, by filling the microplate 18 a,having wells 62 of 5 ml, and the PCR elution tubes 69 of 0.2 ml of themagnetic rack 18 b such that:

-   -   each well of the first row of the microplate 18 a is filled with        the lysed sample comprising the silica particles, hereinafter        the “lysed sample”. The total volume in each well of the first        row is preferably greater than 1.5 ml because of the use of the        5 ml Deepwell microplate and of the volumes handled by the        electronic pipette;    -   each well 66 of the second row of the microplate 18 a is filled        with 1250 μl of washing buffer (e.g. the “NucliSENS easyMAG        Extraction Buffer No. 2” from bioMérieux, bMx reference 280131);    -   each well 66 of the third row of the microplate 18 a is filled        with 1250 μl of washing buffer (e.g. the “NucliSENS easyMAG        Extraction Buffer No. 2” from bioMérieux, bMx reference 280131);    -   each PCR elution tube 69 inserted in the magnetic rack 18 b is        filled with a volume of 100 μl of elution buffer (e.g. the        “NucliSENS easyMAG Extraction Buffer No. 3” from bioMérieux,        reference 280132).

The user then places:

-   -   the electronic pipette 12, with its row of cones 20, in the        housing 38 of the pipette holder 14 in a raised position so as        to allow the introduction of the plate 18 a; and    -   the plate 18 a in the housing 56 of the base 34 with the first        row of wells comprising the lysed sample in line with the cones        20.

The extraction 102 begins with the homogenization of the lysed sample.To do this, the magnetized part 16 is not placed in the base 34 and doesnot therefore interfere with the cones 20. The user turns one of thewheels 50 so as to dip the tips 21 of the cones 20 in the row of wellsof the plate 18 a comprising the lysed sample. The user then selects, bymeans of the interface 24 of the pipette 12, a first pipetting protocolcomprising at least one phase of suction/discharge of the lysed samplein the cones 20, and launches the protocol selected. These phases (e.g.two of them) each comprise at least one suction/discharge cycle (e.g.five cycles), followed by a waiting period of several minutes, forexample 5 minutes. For the purposes of the invention, a suction anddischarge cycle consists in filling at least three quarters of, forexample completely filling, the cones and then in completely emptyingthem, unless specified otherwise by the program.

Once the homogenization is finished, the cones 20 are empty and theirtips 21 dip into the wells containing the lysed sample. The purification102 continues with the capture, in 112, of the silica particles, of thelysed sample, on the internal wall of the cones 20. To this effect, theuser places the magnetized part 16 in the housing 78 of the base 34,selects, by means of the interface 24 of the pipette 12, a secondpipetting protocol and then launches the protocol selected. The secondprotocol comprises a plurality of suction/wait/discharge cycles, e.g.about ten cycles, a suctioning operation being separated from adischarge operation by a few seconds, e.g. about ten seconds. At eachsuctioning operation and each discharge operation, a part of theparticles contained in the lysed sample is captured on the wall of thepipette cones by virtue of the magnetic field produced by the magnetizedpart 16. The magnetic particles, and therefore also their bound nucleicacids, are thus captured in the form of pellets of particles 100 facingthe magnetized part 16, and preferably on a central zone halfway up thecones 20, as illustrated in FIG. 9 .

Once the capture has finished, the lysed sample having been completelydischarged from the cones 20 and the magnetized part 16 still being inposition, the purification 102 continues with a first washing step 114.To this end, the user raises the pipette holder 14 (respectively raisesthe pipette 12) so as to release the plate 18 a from the cones 20,aligns the second row of the plate 18 a with the row of cones 20, thenrepositions the pipette holder (respectively descends the pipette) so asto dip the tips 21 of the cones in the wells of the plate 18 a. The userthen selects, by means of the interface 24, a third pipetting protocolcomprising at least one phase of suction/discharge of the lysed samplein the cones 20, then launches the protocol selected. The third protocolis for example identical to the first protocol. The repeated passing ofthe washing buffer over the particle pellets thus makes it possible towash said particles. This washing step is advantageously completed, orcarried out jointly, with a modulation of the magnetic field capturingthe particles on the cones. For example, the user raises and descendsthe pipette 12, which has the effect of moving the particle pellets onthe cones, or alternatively the magnetized part 16 comprises a set ofpermanent magnets and the user slides the magnetized part 16, in an upand down movement, from its housing 78, such that the intensity and thelines of magnetic fields capturing the pellets vary, while at the sametime maintaining the particles captured on the cones. The modulation ofthe magnetic field thus has the effect of reorganizing the pelletsduring the washing, and increasing the efficiency thereof.

A second washing operation is then carried out in 116 by means of thewashing buffer of the third row of the plate 18 a. For example, thefirst washing buffer is completely emptied out of the cones, then asecond washing operation identical to the first washing operation iscarried out.

Subsequent to this second washing operation, a step of migration 118 ofthe particle pellets 200 into the tips 21 of the cones 20 is carriedout. To do this, the cones 20 preferentially remain filled with thesecond washing buffer in order to facilitate the slide of the pellets200 and remain aligned with the second row of the plate 18 a. The userthen turns one of the wheels 50 so as to raise the pipette 12. Since themagnetized part 16 is rigidly connected to the base 34, the pellets thusremain immobile relative to said part and migrate toward the tips 21 bysliding along the walls of the cones 20 as the pipette is raised. Theuser stops the raising of the pipette 12 once the pellets 200 are in thetips 21, as illustrated in FIG. 10 , at an average distance of a fewmillimeters, e.g. 8 mm, from the open ends of the cones. In thisposition, the user selects and then launches, by means of the interface24, the discharge of the washing buffer contained in the cones 20 intothe wells of the plate 18 a. Optionally, one of the washing phases, oran additional washing phase, consists in removing the magnetized part soas to release the magnetic particles and to carrying out a washingoperation while at the same time stirring the particles in the washingsolution by means of suction and discharge cycles. The particles arethen captured once again by repositioning the magnetized part and byperforming suction and discharge cycles as previously described.

The purification 102 ends with a step 120 of transferring the magneticparticles present in the tips 21 of the cones 20 into the PCR elutiontubes 69. To this effect, the user raises the pipette holder 14, removesthe plate 18 a, places the magnetic rack 18 b in the housing 56 so as toalign the PCR tubes 69 with the row of cones 20, rests the pipetteholder 14 and removes the magnetized part 16 from the base 14 in orderto release the captured magnetic particles from the cones. Once the tips21 have been dipped into the tubes 69, the user selects, by means of theinterface 24, a fourth pipetting protocol, then launches the protocolselected. A first variant of this protocol consists of cycles of suctionand discharge of the elution buffer in the tips 21 of the cones 20,which makes it possible to resuspend the magnetic particles by breakingup the particle pellets. Moreover, the frequency chosen for the cyclesmakes it possible, at each discharge in the tubes 69, for some of themagnetic particles to be captured in the tubes 69 by virtue of themagnetic field of the magnetized part 68 inserted in the rack 64.Furthermore, these cycles make it possible to “rinse” the tips 21 inorder to recover particles adhering to the walls of the cones. In asecond variant of the protocol, suction and discharge cycles are firstof all carried out at a higher frequency so as to stir the buffer andthe particles more vigorously, and therefore to obtain acceleratedhomogenization facilitating the transfer into the elution tubes 69. Thetransferring step ends with the complete discharge of the elution bufferin the tubes 69. Under the effect of the magnetic field of the rack 64,the magnetic particles are then definitively separated from the elutionbuffer, as illustrated in FIG. 11 . The user can thus recover the tubes69 for a subsequent treatment, in particular the elution of the nucleicacids by heating, in a manner known per se.

In the embodiment of the pipette holder previously described, themagnetized part 16 is inserted in the base 34. Thus, when the userwishes to move the plate 18 a forward, he can raise the pipettesufficiently high to perform this operation. This causes, as for themigration of the particles to the tips, the pellets 200 to move over thewalls of the cones 20, which has the advantage of “reorganizing” thepellets which can roll over on themselves. The efficiency of the washingis thus thereby reinforced. On the other hand, this means that the usertakes care not to ever raise the pipette too far, so as not to cause thepellets to leave the cones. To do this, the user can for example raiseor tilt the pipette holder so as to keep the pellets at a distance fromthe cone openings. This option, which requires repeated raising of adevice, the weight of which can be considerable, can however lead in thelong term to musculoskeletal problems. In addition, the user must alsotake care not to raise the pipette holder too much, so that the pelletsdo not leave the cones.

A second embodiment of the pipette holder according to the inventionallows the handling of the plates 18 a, 18 b by raising only thepipette, and therefore avoiding raising the pipette holder 14, while atthe same time guaranteeing that the particle pellets remain at adistance from the tips 21 of the cones. This second embodiment, and alsothe variations generated regarding the method which has just beendescribed, are illustrated in FIGS. 12 and 13 .

More particularly, the second embodiment differs from the firstembodiment by virtue of the means of receiving the magnetized part 16 inthe pipette holder 14. In particular, the base 34 comprises the housing78 for the insertion and removal of the magnetized part 16 as previouslydescribed and the housing 78 is open in its upper part 130 so as to alsoallow the insertion and removal of the part 16 vertically in the housing78. The pipette support 36 also comprises means for attaching themagnetized part 16 in line with the open housing 78, in particular oneor more blocks 132 made of magnetizable material (e.g. made of steel)attached to a rear wall 134 of the mobile pipette support 36 (FIG. 12C).In this way, the magnetized part 16 is rigidly connected to the mobilesupport 36 and remains facing the cones 20 when the user raises anddescends the pipette (in particular during the washing phases), asillustrated in FIGS. 12B to 13A.

In order to carry out the migration of the pellets 200 into the tips ofthe cones 20, the user disconnects the magnetized part 16 from thepipette support 36, by applying a simple downward pressure on the handle78 of the part 16, and raises the pipette 12. The magnetized part 16detaches from the blocks 134, thus remains in the housing 78 of the baseand is therefore rigidly connected to the base 34, inducing migration ofthe pellets 200 into the tips 21 of the cones as previously described(FIGS. 13A and 13B). Once the pipette has been raised, the user replacesthe plate 18 a with the rack 18 b equipped with the PCR elution tubes,removes the magnetized part 16 and redescends the pipette (FIG. 13C,tubes 69 not represented). As a variant, the pipette support 36 cancomprise a housing similar to the housing 78 of the base in which theuser slides the magnetized part, in particular for the washing phases.

A particular extraction method has been described. However, the presentinvention applies to any type of capture of magnetic particles and toany type of pipetting sequence. Likewise, a pipette having 8 channelswith a particular volume has been described. The pipette can compriseany number of channels of any volume depending on the intendedapplication.

In order to increase the number of samples processed, two extractionsystems according to the invention can be coupled, as illustrated inFIG. 14 . For example, the rotation handles 50 can fit together suchthat two extractions can be carried out simultaneously, the user raisingand descending the pipettes 12 at the same time. To this end, thepipettes can also be synchronized, one pipette controlling, for example,the other pipette.

Likewise, a portable and semi-automated extraction system, particularlysuitable for test laboratories having a limited number of extractions tobe carried out daily, has been described. However, the invention can beautomated. For example, the pipette is integrated into an automateddevice which comprises programmable mechanisms for raising anddescending the pipette and for moving the magnet (or foractivating/deactivating electromagnets).

Two washing operations in the wells of the second row and of the thirdrow of the microplate 18 a have been described. However, there can beany number of washing operations. Likewise, a single step of capturingthe particles in the cones has been described. One or more steps ofreleasing the particles, each followed by a further capture step, canalso be provided for.

In order to release the particles, the magnetic field for capturing theparticles is deactivated by removing the magnetized part 16 from itshousing, then cycles of suction/discharge of a buffer are carried out inthe pipette cones so as to detach the particle pellets from the walls ofthe cones and to disaggregate them. Such a procedure takes more than 10minutes to completely detach the pellets from the walls of the coneswith a suction/discharge frequency (complete suction and discharge inthe cones) of 5 cycles per minute. With reference to FIG. 15 , a fasterrelease of a pellet 200 is obtained by carrying out, on the pellet 200,an up and down movement of the meniscus 300 that the buffer 302 forms ina cone 20. In particular, the suction/discharge cycle is regulated suchthat the meniscus 300 travels a limited path 304 on either side of thepellet 200 in order to increase the frequency with which the meniscuspasses over the pellet. Likewise, the frequency of the suction/dischargecycles is increased in order to further increase said frequency ofpassage, in particular a cycle frequency greater than 2 cycles persecond on the zone where the pellet of magnetic particles is present.When this procedure is applied, the pellets detach from the cones inless than 1 minute. The inventors have noted that it is the passing ofthe meniscus over a pellet which helps to detach the latter. Indeed,tests have been carried out by rapidly stirring the buffer in the coneswithout passing the menisci over the pellets (i.e. “simple” movement ofliquid in front of the pellets) without any notable saving of time. Oncethe pellet release phase has been carried out, said phase also havingthe effect of disaggregating the pellets, a phase of stirring bycomplete suction/discharge in the cones is carried out (e.g. 8suction/discharge cycles per minute) so as to finish disaggregating thepellets and to homogenize the buffer comprising the particles. Thesecomplete suction/discharge cycles in the wells of the microplates stir agreater volume, over a longer path, thereby facilitating thehomogenization of the buffer.

A description will now be given of a preferred method for extractingnucleic acids (e.g. DNA and RNA), in particular of viral origin, forexample by means of magnetic silica particles. This method comprises aparticle release phase, e.g. as previously described, followed by aphase of washing in a buffer and of recapturing the particles. A notablesaving of time is obtained, as is an improved extraction. In particular,this method comprises, once the virus lysis step has been carried out:

-   -   1. a first step of capturing the magnetic particles in the        pipette cones, e.g. in the manner previously described;    -   2. followed by a first washing step, and preferably by at least        one second washing step, in different rows of the microplate        that are filled with washing buffer (e.g. the “NucliSENS easyMAG        Extraction Buffer No. 1” from bioMérieux, reference 280130).        Each washing operation comprises cycles of suction/discharge of        the washing buffer with the particles captured on the pipette        cones, and lasts at least 15 seconds, preferably between 25        seconds and 35 seconds, for example 30 seconds, and preferably        less than one minute;    -   3. at least one third washing step in a third row of the        microplate 18 a filled with a washing buffer (e.g. the        “NucliSENS easyMAG Extraction Buffer No. 2” from bioMérieux,        reference bMx 280131). During this third washing step, the        particles are released by removing the magnet, so as to        resuspend the particles, the buffer with the particles in        suspension being suctioned/discharged in the corresponding wells        of the microplate 18 a. The third washing step, preferably        comprising a phase of passing menisci over the pellets as        previously described, lasts a few minutes, in particular 5        minutes;    -   4. a second step of capturing the particles on the pipette        cones, e.g. as previously described;    -   5. optionally, a fourth washing step, the particles being        captured in a third row of the microplate 18 a filled with a        washing buffer (e.g. the “NucliSENS easyMAG Extraction Buffer        No. 2” from bioMérieux, reference bMx 280131);    -   6. a step of migration of the particle pellets into the tips,        followed by a step of transferring into tubes (e.g. comprising        an elution buffer, for example the “NucliSENS easyMAG Extraction        Buffer No. 3” from bioMérieux, reference 280132), e.g. as        previously described.

Washing buffers of the NucliSens range, in particular extraction buffersNo. 1, No. 2 and No. 3, have been described. More generally:

-   -   the extraction buffer No. 1 is a buffer which promotes the        capture of nucleic acids on silica by creating bridges between        the silanol groups of the silica and the phosphate groups of the        nucleic acids. It comprises, for example, guanidinium        thiocyanate, namely a chaotropic agent as described in the        document by R. Boom et al. “Rapid and simple method for        purification of nucleic acids.” Journal of Clinical        Microbiology. 1989; 28 (3): 495-503;    -   the first and second washing operations make it possible to        remove the residual matrix or microorganism debris,    -   the third and fourth washing operations make it possible to        remove the traces of GuSCN and of the inhibitors of a PCR-type        amplification usually subsequently carried out on the DNA/RNA        captured by the magnetic particles,    -   the elution buffer included in the PCR cones makes it possible        to remove any trace of washing buffer and to be under optimal        conditions for the elution step.

The following table compares the results obtained with the deviceaccording to the invention when applying the protocol that has just beendescribed (2 first washing operations followed by a third washingoperation with release of the particles) in comparison with the resultsobtained with a device of the prior art, namely the MiniMag® sold by thecompany bioMérieux and considered to be a reference device in viral RNAextraction. The protocol for the MiniMag® comprises four washing stepswith the washing buffers (two with the “NucliSENS easyMAG ExtractionBuffer No. 1” and two with the “NucliSENS easyMAG Extraction Buffer No.2”). In order to determine the efficiency of the extraction, a real-timePCR amplification (or “q-PCR”) of the lysate extracted is carried outand the Ct (“cycle threshold”, which quantifies a threshold of detectionof nucleic acid in a sample) of each sample is measured. The samplestested in duplicate are samples of 25 grams of raspberry or of greenonion to which is added a solution of Mengo virus which is pure(corresponding to 500 copies of the genome per 25 grams) or diluted to1/10^(th).

Invention (Ct value) MiniMag ® (Ct value) Sample Raspberry Green onionRaspberry Green onion Mengo pure 25.72 26.29 25.87 25.55 24.86 25.061/10 27.87 28.61 28.06 27.95 28.01 27.81 Mengo pure 26.05 26.11 25.8225.87 24.93 24.99 1/10 28.23 28.44 27.59 28.13 28.06 27.76

As can be seen, the extraction of the viral RNA according to theinvention gives results similar to those obtained using the MiniMag®. Inaddition, tests were carried out with various batches of magnetic silicaparticles of diverse quality. It was noted that the extraction accordingto the invention is surprisingly very robust with respect to the qualityof said particles. In particular, tests were carried out on the samesamples with a batch of particles of lower performance grade, theextraction not comprising the release/washing/recapture step aspreviously described. In this case, the degree of extraction was lower.When using the preferred method previously described with the defectiveparticles, results similar to those of the preceding table wereobtained.

An application of the invention to the capture of nucleic acids, e.g.RNA and/or DNA, originating from a lysis carried out before thecapture/washing/migration and transfer phases, has been described. Theinvention also applies to the capture of microorganisms (e.g. bacteria,fungi, yeasts) by means of magnetic particles of which the surface isfunctionalized so as to capture the microorganisms (e.g. covered withphage proteins or with polycations suitable for such a capture in amanner known per se). The magnetic particles with their capturedmicroorganisms are transferred into tubes in order to subsequentlyundergo lysis, for example mechanical lysis. The lysate obtained candirectly be the subject of a treatment, for example a polymerase chainreaction amplification (e.g. a quantitative PCR of q-PCR type), or canbe purified according to the nucleic acid extraction method previouslydescribed.

The invention is particularly suitable for the preparation of amicrobial sample for the purpose of a PCR. Indeed, the sample on whichthe capture of particles in the pipette cones is carried out may have avery large volume (e.g. several milliliters), whereas the final volumeof the tubes into which the particles are transferred can be very small(e.g. less than or equal to 200 microliters, or even less than or equalto 100 microliters). Because of the large volume of the sample, a largenumber of microorganisms are captured. The passage to a very small finalvolume has the effect of concentrating the microorganisms. Thus, theinventors have noted that a single phase of capture from a sample of afew milliliters, followed by a single washing step, is sufficient toobtain results by q-PCR from a lysis carried out in a volume of 5microliters.

In particular, an enrichment of food matrix (chicken aiguillette) withnutritive broth was carried out for 5 h at 41.5° C. A post-contaminationwith a Salmonella Derby strain is carried out at a level of 10² to 10⁴CFU/ml, which corresponds to concentrations that can be reached afterenrichment in the presence of pathogen in the food matrix (i.e.concentrations for which a food batch is determined to be unfit forconsumption). Two procedures were carried out, in duplicate, on eachcontaminated sample, one according to a standardized capture protocolwith the Gene-up® system from bioMérieux, France, and one according tothe invention.

The Gene-up protocol consists of a step of “bead-beating” of the sample(i.e. mechanical disruption of the wall of the bacteria), by taking 20μl of said sample and placing it in a bead-beating tube containing 180μl of washing buffer, followed by shaking for 5 minutes on a microplateshaker for bead-beating. 5 microliters of the final solution are takenand are subjected to a q-PCR.

The method according to the invention consists, for its part, of:

-   -   1. a specific capture step by bringing 2 ml of sample into        contact with a biotinylated phage protein solution (final        concentration 2 μg/ml) by:        -   a. agitating by suction/discharge in the pipette cones for            10 min;        -   b. adding “Hyglos Streptavidin” magnetic particles (50 μl)            and agitating by suction/discharge for 15 minutes (the            bacteria-biotinylated phage protein complexes bind to the            magnetic particles);        -   c. putting in place the magnet for the phase of collecting            the magnetic particles in the cones;        -   d. launching the magnetic particle capture cycle;    -   2. a step of washing in the wells containing the TST (Tris        Saline Tween) washing solution with 5 suction/discharge cycles;    -   3. a step of collecting the magnetic particles in the        5-microliter tubes which are subjected to a bead-beating        treatment, the final solution of 5 microliters then being        subjected to a q-PCR.

The results obtained according to the Gene-up® protocol and according tothe invention are summarized in the table below:

Concentration Invention (Ct value) Gene-up ® (Ct value) 10² CFU/ml 35.2No Ct 35 No Ct 10³ CFU/ml 33.8 No Ct 33.8 No Ct 10⁴ CFU/ml 29.5 34.929.7 No Ct

The estimated gain in sensitivity is 2 log compared with the Gene-upstandard protocol.

The present invention answers a problem of polyvalence for the use ofvarious magnetic capture techniques (nucleic acid purification, magneticimmunoconcentration, etc.). The system according to the invention, whichis evolutive and modulatable, allows:

-   -   steps of capture/washing/elution of magnetic particles to be        carried out using an autonomous system consisting of the        combination of a programmable electronic pipette and of a        support enabling the various abovementioned steps to be carried        out;    -   a number of samples of 1 to 8 to be treated as a function of the        configuration of the electronic pipette used;    -   the samples to be treated in parallel in the context defined        above with a semi-automatic system;    -   2 systems to be combined if required to increase the number of        samples to be treated;    -   it to be possible for the elution steps to be carried out in        various types of tubes: 0.2 ml PCR tubes for recovering the        magnetic silica particles when nucleic acid capture is involved        (e.g. NucliSENS© chemistry) or else bead-beating tubes in the        case of the recovering of magnetic particles having been used        for the recovery of pathogens (magnetic immunoconcentration). To        this end, the system makes it possible to carry out steps of        capture/concentration of pathogens on the magnetic particles and        the in situ lysis thereof by means of ceramic/glass beads (e.g.        method of CapLyse© type).

The invention claimed is:
 1. A method for extracting componentscontained in a biological sample in liquid form, the components beingcapable of binding to magnetic particles, the method comprising: a phaseof mixing the sample with the magnetic particles; a phase of suctioningthe mixture from a well in a tubular pipette cone comprising a tipintended for pipetting liquid; a phase of capturing the magneticparticles on an internal wall of the pipette cone by: applying a firstmagnetic field to the pipette cone, the field being capable ofattracting and holding the magnetic particles in a predetermined zone ofthe pipette cone, termed “capture” zone, above the tip of the cone; andapplying at least one cycle of suction and discharge of the mixturecontained in the pipette cone in a well; at least one phase of washingthe particles captured on the internal wall of the pipette cone by:discharging the mixture contained in the pipette cone; and applying,from a well containing a washing solution, at least one cycle of suctionand discharge of the washing solution in the pipette cone; a phase ofmigration of the magnetic particles on the internal wall of the pipettecone, from the capture zone to the tip of the pipette cone, by carryingout a relative movement of the pipette cone relative to the firstmagnetic field; and a phase of transferring the magnetic particlescontained in the tip of the pipette cone into a recovery well containinga solution, by: deactivating the first magnetic field; releasing themagnetic particles that migrated to the tip of the pipette cone byapplying, from a well containing a washing solution, at least one cycleof suction and discharge of the washing solution in the pipette cone;applying a second phase of capturing the magnetic particles in the tipof the pipette cone by: applying the first magnetic field to the pipettecone, and applying at least one cycle of suction and discharge of themixture contained in the pipette cone in the well containing the washingsolution, placing the tip of the pipette cone in the recovery well; andapplying a second magnetic field from the bottom of the recovery well soas to cause the magnetic particles contained in the tip of the pipettecone to migrate into the recovery well, the second magnetic field beingproduced by a magnet positioned partially or entirely under the tip ofthe pipette cone.
 2. The method as claimed in claim 1, wherein therelative movement comprises moving the pipette cone parallel to alongitudinal axis of the cone, and keeping the first magnetic fieldconstant, the longitudinal axis of the pipette cone remaining at equaldistance from the first magnetic field during the movement of thepipette cone.
 3. The method as claimed in claim 1, wherein the firstmagnetic field applied to the pipette cone is deactivated during theapplication of the second magnetic field.
 4. The method as claimed inclaim 1, wherein the transferring phase comprises the deactivation ofthe first magnetic field followed by the application of cycles ofsuction and discharge of the solution of the recovery well in the tip ofthe pipette cone, the application comprising: a first phase of applyingthe cycles at a first frequency; followed by a second phase of applyingthe cycles at a second frequency, lower than the first frequency.
 5. Themethod as claimed in claim 1, comprising, prior to the capturing phase,a phase of stirring the mixture contained in the pipette cone byapplying at least one cycle of suction and discharge of the mixture inthe pipette cone.
 6. The method as claimed in claim 1, wherein therelease of the magnetic particles comprises a phase of applying thecycles in such a way as to carry out an up and down movement of ameniscus of the solution over a pellet of particles captured in thepipette cone, the up and down movement of the meniscus being carried outon a portion of the cone less than the total length of the pipette cone.7. The method as claimed in claim 6, wherein the release of the magneticparticles comprises a second phase of applying the cycles so as tototally suction and discharge the cone washing solution.
 8. The methodas claimed in claim 7, wherein the frequency of application of thecycles of the second phase is lower than the frequency of application ofthe cycles of the first phase.
 9. The method as claimed in claim 1,wherein, prior to the release of the magnetic particles, at least twowashing phases are carried out in two distinct washing solutions. 10.The method as claimed in claim 1, wherein the components contained inthe biological sample are nucleic acids.
 11. The method as claimed inclaim 1, wherein the components contained in the biological sample aremicroorganisms, and wherein the method comprises a single capturingphase and a single washing phase.
 12. The method as claimed in claim 11,wherein the mixture of the sample with the magnetic particles has avolume of greater of 1 milliliter, and wherein the volume of therecovery well is less than 200 microliters.
 13. The method as claimed inclaim 1, comprising, prior to the transferring phase, at least one phaseof washing the particles captured on the internal wall of the pipettecone: by suctioning the washing solution in the pipette cone; then bymodulating the first magnetic field applied to the magnetic particles inorder to capture the particles on the internal wall of the pipette cone;then by discharging the pipette cone washing liquid.
 14. The method asclaimed in claim 13, wherein the modulation of the first magnetic fieldis carried out: by moving the pipette cone parallel to a longitudinalaxis of the cone, and by keeping the first magnetic field constant;and/or by passing magnets spaced out from one another in front of thecaptured particles.
 15. The method as claimed in claim 1, wherein thevolume of the pipette cone is at least ten times greater than the volumeof the recovery well.
 16. The method as claimed in claim 1, wherein thevolume of the mixture is at least three times greater than the volume ofthe pipette cone.
 17. The method as claimed in claim 1, wherein thecomponents are selected from the group consisting of nucleic acids,microorganisms, proteins, and peptides.
 18. The method as claimed inclaim 1, wherein the pipette cone comprises an open end proximate to thetip.
 19. The method as claimed in claim 1, wherein a pipette is equippedwith the pipette cone and the pipette is inserted into a pipette supportof a pipette holder.
 20. The method as claimed in claim 19, wherein thepipette support is translationally mobile relative to a base of thepipette holder.
 21. The method as claimed in claim 1, wherein the phaseof capturing the magnetic particles on the internal wall of the pipettecone comprises applying multiple cycles of suction and discharge of themixture contained in the pipette cone in the well.
 22. The method asclaimed in claim 1, wherein the at least one phase of washing theparticles captured on the internal wall of the pipette cone is performedwhile applying the first magnetic field to the pipette cone.
 23. Themethod as claimed in claim 22, wherein during the at least one phase ofwashing the particles captured on the internal wall of the pipette cone,the pipette cone is moved relative to the first magnetic field, therebyreorganizing the magnetic particles on the internal wall of the pipettecone.